Luciferase from Vibrio campbellii is more thermostable and binds reduced FMN better than its homologues y Chutintorn Suadee 1 , Sarayut Nijvipakul 1 , Jisnuson Svasti 1 , Barrie Entsch 2,3 , David P. Ballou 2 and Pimchai Chaiyen 1, * 1 Department of Biochemistry and Center for Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand; 2 Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, 48109-06060, USA; and 3 School of Biological, Biomedical and Molecular Sciences, University of New England, Armidale, NSW, 2351, Australia Received June 8, 2007; accepted August 11, 2007; published online August 30, 2007 A new luciferase from V. campbellii (Lux_Vc) was cloned and expressed in Escherichia coli and purified to homogeneity. Although the amino acid sequences and the catalytic reactions of Lux_Vc are highly similar to those of the luciferase from V. harveyi (Lux_Vh), the two enzymes have different affinities toward reduced FMN (FMNH  ). The catalytic reactions of Lux_Vc and Lux Vh were monitored by stopped-flow absorbance and luminescence spectroscopy at 48C and pH 8. The measured K d at 48C for the binding of FMNH  to Lux_Vc was 1.8 kM whereas to Lux_Vh, it was 11 kM. Another difference between the two enzymes is that Lux_Vc is more stable than Lux_Vh over a range of temperatures; Lux_Vc has t 1/2 of 1020 min while Lux_Vh has t 1/2 of 201 min at 378C. The superior thermostability and tighter binding of FMNH  make Lux_Vc a more tractable luciferase than Lux_Vh for further structural and functional studies, as well as a more suitable enzyme for some applications. The kinetics results reported here reveal transient states in the reaction of luciferase that have not been documented before. Key words: bioluminescence, flavin, luciferase, monooxygenase, Vibrio campbellii. Abbreviations: C 1 , reductase component of p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii; FMNH  , reduced form of FMN; HPA, p-hydroxyphenylacetate; K d , dissociation constant; k obs , apparent rate constant; Lux, luciferase; Lux_Vc, luciferase from V. campbellii; Lux_Vh, luciferase from V. harveyi; Lux:FMN, complex of luciferase and oxidized FMN; Lux:FMNH  , complex of luciferase and reduced FMN. Bacterial bioluminescence is a phenomenon caused by luciferase (Lux), an enzyme catalyzing the reaction of O 2 with reduced FMN and a long-chain aliphatic aldehyde, and resulting in oxidized FMN, water, and a fatty acid, with concomitant emission of blue–green light (Fig. 1) (1, 2). This reaction has been found mainly in the three genera, Vibrio, Photobacterium, and Xenorhabdus, and the enzymes from only five species have been isolated and studied: Vibrio(Photobacterium) fischeri, Vibrio(Beneckea) harveyi, Photobacterium phos- phoreum, Photobacterium leiognathi, and Photorhabdus (Xenorhabdus) luminescens (3–5). The first four species are found in marine environments, while the last species is found in terrestrial habitats (6–9). All of these enzymes catalyze the same chemical reactions and are hetero- dimers composed of an a subunit with a molecular mass of 40–52 kDa and a b subunit with a molecular mass of 36–41 kDa (1, 6, 10–16). The amino acid sequences of all these enzymes are similar (50–60% identity). However, the kinetics of light decay among luciferases from different species varies, with the luciferase from V. harveyi (Lux_Vh) having the slowest light decay kinetics (0.12 s 1 at 308C). The rest have considerably faster rates of decay of 1s 1 at 308C(17–19). Variation has also been found in the spectrum of light emitted from different bacterial strains. In V. fischeri strain Y-1, a yellow fluorescent protein (YFP) was reported as an accessory protein that formed a transient complex with Lux and shifted the emitted light to longer wavelengths (17, 20–22). A similar accessory protein called lumazine was isolated from P. phosphoreum and P. leiognathi. In contrast to YFP, lumazine actually shifts the emitted light to shorter wavelengths (23–25). Studies have shown that FMNH  is the first substrate binding to Lux, and then oxygen reacts rapidly with the bound FMNH  to form a C(4a)–peroxyflavin (C(4a)– FMNHOO  )(26–29). In the absence of aldehyde sub- strate, this intermediate slowly eliminates H 2 O 2 to yield FMN with no emission of visible light (26–27). When present, aldehyde binds and reacts with the bound C(4a)- FMNHOO – to form a flavin–C(4a)–peroxyhemiacetal adduct (C(4a)–FMNHOOR), which then decomposes to form the carboxylic acid product and an excited flavin intermediate that emits blue–green light (30, 31). The species emitting light was shown to be an excited state C(4a)–hydroxyflavin (C(4a)–FMNHOH) (30, 32), which, after emitting light, dehydrates to yield FMN. It should be noted that the fluorescence quantum yield of y Nucleotide sequence data reported are available in the GenBank database under the accession number EF394780. *To whom correspondence should be addressed. Tel: +662-201- 5607, Fax: +662-354-7174, E-mail: scpcy@mahidol.ac.th J. Biochem. 142, 539–552 (2007) doi:10.1093/jb/mvm155 Vol. 142, No. 4, 2007 539 ß 2007 The Japanese Biochemical Society.